The lack of oestrogens in post-menopause causes alterations to the vaginal epithelium in more then 50% of women aged between 50 and 60 years [Pandit et al, 1997]. These disorders are characterised by their chronicity and recurrence, sometimes also accompanied by consequent sexual dysfunction, and their significance easily surpasses their actual severity in as much as they reinforce the fear of a general decline [Rossin-Amar, 2000]. The lack of oestrogens causes progressive atrophy of the vulva, which results in a gradual breakdown of the mucus, with 30-50% reduction of vascularisation levels. The simultaneous reduction of glycogen, in particular in cells of the intermediate stratum, causes a change to the vaginal ecosystem, with a consequent increased risk of the presence of pathogenic agents responsible for recurrent vaginitides. In addition, vaginal atrophy can also occur in pre-menopausal women who have undergone a surgical intervention due to endometrial cancer, including pelvic cancer, or paraaortic lymphadenectomy and are treated with adjuvant vaginal brachytherapy (VBT) in order to reduce the risk of local relapses and to improve the survival rate of patients [Scholten 2005; Keys 2004; Lee 2006]. All of these treatments can give rise to adverse effects in a variable percentage of patients, including dysuria, vaginal pain, atrophy of the vaginal mucus, and vaginal dryness [Dickler 2010]. These symptoms can be considered a significant health problem for a considerable part of the global female population, since they are often linked to inflammation, dyspareunia and loss of sexual enjoyment.
The loss of oestrogens is recognised as the most common cause of the symptoms of the menopause, often causing the treatment of post-menopausal vulvo-vaginal disorders with systemic hormone replacement therapy (HRT). However, the involvement of oestrogens in the genesis and progression of tumours of the endometrium, ovary and breast, even though the increased risk of cancer depends on the type of hormone replacement therapy, the duration of use, body mass and the period between menopause and the onset of the hormone replacement therapy, leads to scepticism among women and also to reluctance among some doctors to advise the adjuvant therapy [Hendrix et al, 2005; Grodstein et al, 2004], which has been placed seriously in doubt. Until now, no agreement has been reached in respect of the suitable therapy: HRT must be administered to women suffering from menopausal disorders in order to satisfy their individual needs, taking into account their individual risk profile and the general therapeutic objectives.
For these reasons, formulations of intravaginal oestrogens have been introduced in order to avoid systemic exposure to oestrogens and have been preferred in women who have no other menopausal symptoms requiring systemic treatment [Johnston et al, 2004; Willhite and O'Connell, 2011]. However, it has been demonstrated that topical oestradiol is significantly absorbed and appears in the general circulation [Martin et al, 1979; Furuhjelm et al, 1980; Deutsch et al, 1981; Mandel et al, 1983; Ballag, 2005; Kendall et al, 2006; Kvorning and Jensen, in: Publication Presented at: International workshop, Copenhagen, 1986], thus confirming the exposure to the increased risk of breast cancer and endometrial cancer after local hormone treatment [Notelovitz et al, 2002; Rioux et al, 2000; Schiff et al, 1977]. In light of this consideration, non-hormonal preparations have been developed for the treatment of vaginal atrophy [Kendall et al, 2006; Berger et al, 2008], such as isoflavones derived from soy. However, the use of topical formulations based on soy isoflavones for the purpose of preventing post-menopausal symptoms has provided insufficient results in terms of efficacy [Levis et al, 2011].
The oestrogen receptor alpha (ERα) performs its function predominantly via binding to its ligand, 17β oestradiol (E2), which induces conformational changes that allow the recruitment of coactivator molecules and the binding to oestrogen response elements (EREs) on the DNA to control the transcription of target genes (“classic” signal pathway of the ER). In the last decade, emerging tests have supported the importance of an alternative signal pathway of ERα (referred to as a “non-classic” path) in mediating the actions of oestrogens [Hall et al, 2001].
This path can bring genotropic effects in target cells and is independent of the binding between ER and ERE. The genotropic signal mediated by ERα involves other transcription factors, such as the activator protein 1 (AP1), the specificity protein 1 (SP1), and NF-κB [Paech et al, 1997; Coleman and Smith, 2001; Porter et al, 1996; Cerillo et al, 1998], which in turn mediate the regulation of the transcription of their target genes. The genotropic signal mediated from ERα includes the activation of the membrane-associated receptor and the stimulation of cytoplasmic pathways, such as PI3K/AKT, ERK and the signalling cascade of p38 [Singh, 2001; Watters et al, 1997; Zhou et al, 1996]. It is interesting to note that the factors determining whether the signal mediated from ER passes via the classic pathway or via the non-classic pathway, remain virtually unknown.
In recent years, it has been demonstrated that various growth factors play a role in the proliferation of cancerous cells of the breast, interacting with the pathway of the oestrogens, although the mechanisms and the effects of such interaction are not yet clear. A certain number of studies have produced suggestions of a cross-talk between the signalling of ER and the signalling of EGF/EGFR. In particular, it has been hypothesised that the development of resistance to tamoxifen in breast cancer cells can be correlated with the ER-mediated activation of EGFR, HER2/neu and IGFR.
Keratinocyte growth factor (KGF/FGF7) (NCBI Reference Sequence NM_002009.3, GenBank amino acid sequence: CAG46799.1), a member of the family of fibroblast growth factors (FGFs), plays a fundamental role in the regulation of cell proliferation, migration and differentiation during development and in response to damage and repair of tissues [Finch and Rubin, 2004]. It acts by binding to the receptor tyrosine kinase FGFR2-IIIb/KGFR, generated by means of alternative splicing of the FGFR2 gene and expressed predominantly on the epithelial cells of various organs, playing a key role in the control of epithelial growth and differentiation [Zhang et al, 2006]. The KGF/KGFR pathway is essential to maintain the integrity and function of adult epithelial cells due to the cytoprotective and regenerative effect of KGF. In fact, the expression of KGF is strongly over-regulated after lesions in various epithelial tissues, such as skin, kidneys, bladder, pancreas, stomach and intestine [Werner et al, 1992; Marchese et al, 1995; Brauchle et al, 1996; Werner, 1998]. In addition, KGF protects against lesions of the pulmonary epithelium and improves the distal repair of the airways, stimulating cell proliferation, inhibiting apoptosis and the free oxygen radicals, and mobilising epithelial progenitor cells [Gomperts et al, 2007].
It has been demonstrated that treatment with recombinant KGF (palifermin) is able to protect epithelial cells against a variety of lesions, including damage induced by radiation, and KGF has therefore been approved by the FDA for the treatment of severe oral mucositis resulting from the radiotherapy and/or chemotherapy of cancer in patients with haematological or head or neck tumours [Spielberger et al, 2004; Beaven and Shea, 2007; Brizel et al, 2008; Barash et al, 2009]. On the other hand, the administration of KGF to mice with developing vaginas during the neonatal period results in oestrogen growth, independently of the vaginal epithelium, thus suggesting a potential link between treatment with oestrogens and activation of the signal of KGF/KGFR [Masui et al, 2004]. In addition, the authors have already carried out the first autologous transplant of vaginal tissue in a woman suffering from Mayer Rokitansky Kuster Hauser syndrome (MRKHS) by means of the use of autologous vaginal tissue cultivated in vitro [Panici et al, 2007], thus forming the ideal model in vitro to analyse the effect of oestrogens and KGF on the trophism of the vaginal mucus.
International application WO 2006/083087 describes that the N-terminal peptide AIMP1 is able to stimulate synthesis of collagen and/or expression of KGF. However, the ability of the peptide to pass through the epidermal layer and the plasma membrane and to therefore be truly effective in cutaneous treatments has not been demonstrated. As is instead known in literature, the protein substances do not pass through the epidermal layer in the presence of undamaged skin.
There is thus a need to provide a therapeutic agent able to locally treat menopausal disorders, including vaginal dryness and atrophy, substituting the therapies already in use (formulations based on oestrogens).